Molecular and Cellular Biochemistry 170: 1–8, 1997. © 1997 Kluwer Academic Publishers. Printed in the Netherlands.
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Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells Ghassan Bkaily, Doris Jaalouk, George Haddad, Nadine Gros-Louis, May Simaan, Radha Naik and Pierre Pothier MRCC Group on immuno-cardiovascular interaction, Department of Anatomy and Cell Biology, Faculty of Medicine Université de Sherbrooke, Sherbrooke, Quebec, J1H 5N4, Canada Received 12 December 1995; accepted 29 March 1996
Abstract The effect of taurine on the different types of ionic currents appears to depend on [Ca] o and [Ca]i and may also vary accordingly to tissue or cell type studied. Using microfluorometry and Ca2+ imaging techniques, short-term exposure (5–10 min) of single heart cells to taurine was found to increase total intracellular free Ca2+ in a concentration-dependent manner. However, long-term exposure of heart myocytes to taurine was found to decrease both nuclear and cytosolic Ca2+ without significantly changing either nuclear or cytosolic Na+ levels, as measured by 3-dimensional Ca2+ and Na+ confocal imaging techniques. Longterm exposure to taurine was found to prevent cytosolic and nuclear increases of Ca2+ induced by permanent depolarization of heart cells with high [K]o. This preventive effect of taurine on nuclear Ca2+ overload was associated with an increase of both cytosolic and nuclear free Na+. Thus, the effect of long-term exposure to taurine on intranuclear Ca2+ overload in heart cells seems to be mediated via stimulation of sarcolemmal and nuclear Ca2+ outflow through the Na+-Ca2+ exchanger. (Mol Cell Biochem 170: 1–8, 1997) Key words: taurine, heart cells, calcium, sodium, confocal microscopy, nucleus, fluo-3, sodium green, Ca2+ overload
Introduction Taurine (2-aminoethylsulfonic acid) is the second most abundant amino acid after glutamate and one of the most intriguing amino acids in the body. Although it is well known that the intracellular concentration of taurine in cardiac myocytes is very high (millimolar range) and even higher than that in plasma, its role is not fully understood [25]. In the heart, taurine deficiency produces dilated cardiomyopathy [19] and electrophysiological abnormalities [17]. Taurine antagonises the inotropic actions of both high and low Ca2+ concentrations on the heart [22, 28] and protects against the Ca2+ paradox [26, 28]. Review of taurine action on ionic currents reveals that the action of this amino acid on the L-type calcium and the fast Na+ channels is complex and does not explain the positive inotropic effect of taurine nor its protective effect against calcium overload [2, 3, 6, 20–28]. More-
over, virtually all studies regarding taurine effects on L-type and T-type Ca2+ currents as well as the fast Na2+ currents have been done using short-term exposure to high concentrations of taurine (10–40 nM) [20–24]. To our knowledge, there are no studies showing the long-term effect of this amino acid on intracellular free Ca2+ and Na+ which may explain in part its cardio-protective effect against Ca2+ overload. Recently in our laboratory, using confocal microscopy, we reported implication of the nucleus in cytosolic Ca2+ buffering of heart cells [8]. In this study, using microfluorometry and laser confocal microscopy, we tested the effect of short and longterm exposure of taurine on the total intracellular level ([ ]i) as well as on the level of cytosolic ([ ]c ) and nuclear ([ ]n) free Ca2+ and Na+ of normal and depolarized single heart cells of chick embryo. Our results suggest that the observed effect of taurine on total [Ca]i and [Na] i is mainly nuclear and dependent on the time of exposure to the amino acid. Further-
Address for offprints: G. Bkaily, Department of Anatomy and Cell Biology, Université de Sherbrooke, Faculty of Medicine, Sherbrooke, Quebec, J1H 5N4, Canada
2 more, taurine prevents but does not reverse intracellular Ca2+ overload induced by sustained depolarization of the cell membrane of heart cells.
Materials and methods Isolation of chick ventricular myocytes Ventricular myocytes were obtained from the lower third of the heart of ten-day-old embryonic chicks as described previously [4–13]. Briefly, single cell preparations were harvested by repeated dispersions in sterile Hank’s minimum essential medium (HMEM) containing 0.1% trypsin and 1.8 mM Ca 2+. Following centrifugation and washing, cells were resuspended in culture medium consisting of HMEM supplemented with 5% fetal bovine serum and 50 IU/ml penicillinG-potassium (Ayerst, Toronto). All other culture solutions were purchased from Gibco (St Louis, MO). Cultured cells were maintained at 37°C in 5% CO2, 95% air and used after 1–24 h in culture.
Loading of Fura 2/AM for microfluorometry Isolated myocytes were cultured on 25 mm glass coverslips which formed the bottom of the experimental bath chamber [5, 7, 8]. Cells were loaded with the fluorescent ratiometric calcium indicator fura-2/AM (Calbiochem, La Jolla, CA) according to the method described previously [7, 8]. Prior to loading, coverslips were washed 3 times in Tyrode’s solution (Sigma, St Louis) containing 10 mM NaHCO 3, 5 mM HEPES, 1.8 mM CaCl2. 1 mM MgCl2, 2.7 mM KCl, 137 mM NaCl, 3.6 mM NaH2PO4 and 5.5 mM D-glucose (buffered to pH 7.4 with Tris base). The osmolarity of the Tyrode’s solution with or without 0.1% bovine serum albumin (BSA) was adjusted to 310 mOsm with sucrose. Myocytes were incubated with freshly prepared Fura-2/AM (1 µm final concentration in Tyrode-BSA solution) for 30 min at 28°C [7]. Stock solutions of the probe were prepared by addition of DMSO to 1 mM frozen aliquots. After loading, the cells were washed with Tyrode’s solution and incubated for a further 30 min at 28°C in order to ensure complete hydrolysis of acetoxymethyl ester groups. The cells were again washed in Tyrode’s solution prior to microfluorometric measurements. Microfluorometric measurements In order to monitor total intracellular Ca2+ changes of chick myocytes, fluorescence measurements were made using the double excitatory wavelength method (340/380 ratio) using a Deltascan and Imagescan microfluorometer (Photon Tech-
nology International Inc., Princeton, NJ) equipped with a NEC Power Mate 386/20 and accompanying PTI software enabling instrument control, data acquisition and analysis [7]. Calculation of intracellular Ca2+ was performed using the standard Grynkiewicz equation [15, 29] included in the software [7], in which [Ca 2+]i = KD (F – Rmin/Rmax – F), where the KD for fura-2 is 224 nM, F is the experimental fluorescence value, R max is the maximal fluorescence of fura-2 in the presence of saturating Ca2+ , and Rmin is the minimal fluorescence in the presence of minimal Ca2+ . Rmax and Rmin were determined at the end of each experiment using the divalent cation ionophore ionomycin (2 × 10–5 M) to permeabilize the cell (R max) and 30 mM of the Ca2+ chelator EGTA (Rmin). Loading of Fluo-3-AM or sodium Green-AM for confocal microscopy Cells were loaded according to the method described elsewhere [8]. Isolated ventricular myocytes were cultured and mounted in the same manner as cells for Fura-2/AM measurements. Frozen stocks of Fluo 3-AM or Sodium Green-AM (Molecular Probes OR) were reconstituted in DMSO and diluted to a final concentration of 13.5 µM in Tyrode’s-BSA. The cells were incubated for 45 min at room temperature, washed, and further incubated for 15 min at room temperature to complete hydrolysis of acetoxymethyl ester groups. Pluronic acid was added to the initial Sodium Green preparation in order to facilitate cell loading.
Ca 2+ and Na+ imaging using confocal microscopy Confocal microscopy Fluo-3 or Sodium Green loaded cells were examined with a Molecular Dynamics (Surmyvale, CA) Multi Probe 2001 confocal argon laser scanning (CSLM) system equipped with a Nikon Diaphot epifluorescence inverted microscope and a 60X (1.4 NA) Nikon Oil Plan achromat objective. The 488 nm argon laser line (9.0 mV) was directed to the sample via a 510 nm primary dichroic filter and attenuated with a 3% neutral density filter to reduce photobleaching. Pinhole size was set at 100 µm. The image size was 512 × 512 pixels with a pixel size of 0.11 µm. Laser line intensity, photometric gain, PMT settings and filter attenuation were kept constant throughout the experimental procedures [8]. Calcium and sodium fluorescence studies Changes in cytosolic and intranuclear calcium and Na+ fluorescence upon addition of increasing concentrations of taurine to the external Tyrode’s medium was measured in Fluo-3 (for Ca 2+) and Sodium Green (for Na+) loaded myocytes [8]. For short-term treatment with taurine, cells were scanned prior
3 to and after addition of taurine to monitor cell response to the drug. Serial optical scans were performed 2–10 min after addition of each taurine concentration. A total of 12–15 scans (512 × 512) were performed for each series with a step size of 0.8 to 1.0 µm, although the number of sections and step size were rigorously maintained during the course of each experiment in order to localize calcium or sodium variations within the cytosol and the boundaries of the nucleus. For long-term treatment, the cells were scanned after a 24 h exposure to taurine alone or after pretreatment with high potassium (30 mM) in Tyrode’s solution in the absence or presence of taurine. Nuclear staining At the end of each experiment, the nucleus was stained with 100 nM of live cell nucleic acid stain Syto 11 (Molecular Probe, Oregon U.S.A.) according to method described previously [8]. In brief, serial optical scans were taken immediately after development of the stain (approximately 8–10 min) while maintaining positioning, number of sections and step size identical to that used for calcium or sodium staining. 3D reconstructions of the nucleus [8] were performed through volume rendering and used as templates to delineate nuclear from cytosolic free ion (Fig. 5). Volume rendering and nuclear calcium measurements Scanned images were transferred onto a Silicon Graphics Indy 4000 workstation equipped with Molecular Dynamics’ Imagespace analysis and volume workbench software modules. Reconstruction of 3-D images were performed on Gaussian-filtered serial sections and are represented as closest intensity projections for calcium distribution and lookthrough extended-focus projections for both nucleus and calcium co-localisation studies [8]. Images are represented as pseudo-coloured representations according to an intensity scale of 0 to 255 with lowest intensity in black and highest intensity in white (Fig. 5). Measurement of calcium or sodium uptake within the nucleus was performed on 3-D reconstructs (section series). The nuclear area following Syto 11 staining was isolated from the rest of the cell by setting a lower intensity threshold filter to confine relevant pixels. A 3-D binary image series of the nuclear volume was then generated for each cell using the exact same x, y and z set planes as those used during calcium or sodium uptake. By then applying these binary image patterns to the same cell but labelled for calcium or sodium (the binary image serves as a ‘cookie cutter’), a new 3-D projection was created depicting fluorescence intensity levels exclusively within the nucleus. Hence, by ‘removing’ the nucleus from the surrounding cytoplasm, we were then able to measure mean calcium or sodium intensity values in the entire nuclear volume while eliminating possible contribution of perinuclear calcium or sodium to our measurements [8].
Statistics Intranuclear free calcium or sodium intensity levels are represented either as mean fluorescence intensity values or as the percentage of increase relative to control levels. Values are expressed as means ± SEM. Statistical significance was determined using the analysis of variance (ANOVA) test followed by either a Tukey-Kramer or Dunnett’s multiple comparison test to assess statistical significance of the results. p values of less than 0.05 were considered as significant.
Results Effect of short-term treatment with taurine on [Ca]i , [Ca]c and [Ca]n Using Fura-2 calcium microfluorometry technique [4, 5, 7, 8], we tested the effect of short-term exposure (10–20 min) to taurine (1–80 mM) on the level of total intracellular free Ca2+ ([Ca]i ) in single ventricular heart cells of 10 day-old embryonic chick. Short-term exposure to 1 mM (Fig. 1B) of taurine had no significant effect on [Ca]i. Increasing the concentration of taurine up to 5 and 10 mM significantly increased the sustained level of [Ca]i compared to control levels (Figs 1C–D). Further increases of extracellular taurine up to 20, 40 and 80 mM induced, in a concentration-dependent manner, a further rise in the sustained level of [Ca]i which was highly significant (Figs 1F–G) when compared to control levels. Figure 1 summarizes the effect of short-term exposure to different concentrations of taurine on total intracellular free calcium in chick heart cells. In order to determine the localization of the intracellular Ca2+ induced by taurine, steady-state cytosolic ([ ]c) and intranuclear free concentrations ([ ]n) of Ca2+ ion were measured in isolated ventricular cells from 10-day-old embryonic chick heart by Fluo3, three-dimensional confocal microscopy technique [4, 5, 7, 8]. As can be seen in Fig. 1H, the steady-state resting basal level of [Ca]n in heart cells was higher than that in the cytosol. These results with [Ca]n and [Ca]c are similar to those recently reported by our laboratory for single heart cell preparations [8]. Shortterm exposure to a low concentration of taurine (1 mM) had no significant effect on both steady-state [Ca]n and [Ca]c, (Fig. 1I). Increasing the concentration of taurine up to 20 mM increased both steady-state cytosolic and nuclear free Ca2+ although significantly only in the nucleus (Fig. 1J). Effect of short-term treatment with taurine on [Na]c and [Na]n In another series of experiments, the effect of short-term exposure to taurine on steady-state levels of [Na]c and [Na]n
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Fig. 1. Effect of taurine on total, as well as cytosolic and nuclear [Ca]i in isolated heart cells. A: Total basal intracellular free Ca2+ in 10-day-old embryonic chick single heart cells using Fura-2 microfluorometry. B: Addition of 1 mM taurine slightly increased the sustained level of [Ca] i. C–G: Increasing the concentration of taurine from 5–80 mM increased the sustained [Ca] i in a dose-dependent manner. H: Basal relative nuclear and cytosolic Ca2+ fluorescence using Fluo-3 three-dimensional measurement of free Ca2+ fluorescence intensity. I: 3-D measurement of steady-state effect of short-term exposure to 1 mM taurine on cytosolic and nuclear Ca2+ fluorescence intensity. J: Significant increase of nuclear Ca2+ fluorescence intensity by short-term exposure to 20 mM taurine. Serial optical images were recorded after each sequential addition of taurine to the extracellular medium. Mean fluorescence intensity values were calculated from the nuclei and cytosol of 3-D reconstructed cells as described in the methods section. The data are expressed as absolute free Ca 2+ values or in terms of mean relative free Ca 2+ fluorescence intensity. Values are represented as means ± SEM with the number of experiments indicated in brackets. *p < 0.05, **p < 0.01.
were done using the Na+ fluorescent probe Sodium Green and 3D-confocal microscopy technique. As can be seen in Fig. 2, the steady-state level of [Na] n was higher than that in the cytosol (Fig. 2A). Short-term treatment with 1 mM taurine slightly increased both [Na] n and [Na]c (Fig. 2B). Increasing the concentration of taurine up to 20 mM further increased [Na]n and [Na]c although only significant in the nucleus. (Fig. 2C).
Effect of long-term treatment with taurine on cytosolic and nuclear Ca2+ and Na+ levels The effect of long-term treatment (24 h) with taurine (1, 20 and 80 mM) was also performed using Fura-2 Ca2+ whole cell imaging technique. As can be seen in Fig. 3, long-term treatment with 1 mM taurine significantly (p < 0.01) decreased total intracellular free Ca2+ [Ca] i of single heart cells. Longterm pretreatment with 20 and 80 mM taurine did not further decrease [Ca]i levels. Three-dimensional cytosolic ([ ]c ) as well as intranuclear concentrations ([ ]n) of both free Ca2+ and Na+ ions in single 10 day-old chick ventricular heart cells were measured by confocal microscopy using Fluo-3 and Sodium Green respectively [5, 8]. As can be seen in Fig. 4, the resting basal levels of [Ca] n and [Na]n in heart cells were higher than that in the cytosol (Figs 4A and F). Figure 5A shows a sagittal and crosssectional view of a 3D-reconstructed isolated ventricular myocyte illustrating the intracellular (cytosolic and nuclear)
Fig. 2. Effect of short-term exposure to taurine on 3-D cytosolic and nuclear [Na] in single heart cells. A: Basal nuclear and cytosolic Sodium Green fluorescence intensity of 10 day-old embryonic chick heart cells. B: Short-term exposure to 1 mM taurine had no significant effect on both [Na]c and [Na]n. C: Short-term exposure to 20 mM taurine induced a significant steady-state increase of [Na] n but not of [Na]c. The results are expressed in terms of mean relative free Na + fluorescence intensity. Values are expressed as means ± SEM. Number of experiments is indicated in brackets. *p < 0.05.
distribution of Ca2+. Prolonged exposure (12–24 h) to a low concentration of taurine (1 mM) significantly decreased both [Ca]n and [Ca]c (Figs 4B and 5D). Figure 5D shows a sagittal and cross-sectional view of a 3D-reconstructed isolated
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Discussion
Fig. 3. Long-term effect of taurine on total [Ca]i in chick embryonic heart cells using Fura-2 Ca2+ imagery. Long-term treatment (24 h) to 1 mM taurine significantly decreased [Ca]i (***p < 0.001). Increasing extracellular taurine up to 20 and 80 mM did not further decrease [Ca]i. Results are expressed in terms of intracellular Ca2+ concentration. Vertical bars represent means ± SEM with the number of experiments indicated in brackets.
heart cell in which the intracellular (cytosolic and nuclear) distribution of Ca2+ after 24 h treatment with 1 mM taurine is illustrated. [Na]n was also decreased with long-term treatment with taurine without any significant change in [Na]c (Fig. 4G). In order to induce Ca2+ overload, membranes of single heart cells were depolarized by increasing extracellular K+ from 5–30 mM [5, 7]. The cells were exposed to 30 mM [K]o for 12–24 h. As shown in Figs 4C and 5B, long-term sustained depolarization of the cell membrane with high extracellular K+ significantly increased the steady-state [Ca]n without affecting the level of free cytosolic Ca2+ ; however there were no changes in either [Na]n or [Na] c with permanent long-term depolarization of heart cells (Fig. 4H). Inducing nuclear Ca2+ overload with long-term permanent depolarization of the cell membrane (24 h) followed by (not shown) or together with long-term exposure to 1 mM taurine in presence of high [K]o generated a further increase of [Ca]n (Figs 4D and 5C), along with an apparently non significant decrease of [Na]n (Fig. 4I). There was no change in cytosolic levels of either free Ca2+ or free Na +. Long-term pre-treatment with 1 mM taurine (12–24 h), followed by long term depolarization (12 h) of cell membranes with high extracellular K+ in the presence of taurine prevented the increase of [Ca]n induced by long term depolarization alone (Figs 4E and 5E). On the other hand, both cytosolic and nuclear free level of Na+ were significantly increased (Fig. 4J).
Using three-dimensional confocal microscopy, Fluo-3 Ca2+ measurements showed that the basal cytosolic free Ca2+ level in ventricular heart cells of 10-day-old chick embryo is lower than that of the nucleus. These results are in accordance with that recently reported by our group [8] and others [1, 15, 18]. Using 3-D cytosolic and nuclear Ca2+ laser confocal microscopy, our results show that the total intracellular free Ca2+ measured in resting cells using classical fura-2 microfluorometry is in fact partly nuclear. Thus, an increase or decrease of total [Ca]i in response to a specific compound using Fura-2 (or other fluorophores) could be mainly nuclear and/ or cytosolic. The same approach, using the sodium fluorescent probe, Sodium Green, also showed that basal cytosolic free Na+ in ventricular cells is lower than that of the nucleus. These results suggest a possible role of the nucleus in cytosolic Na+ buffering. The mechanism by which Na + crosses the nuclear membrane as well as its nuclear physiological role are not known. Our results also showed that short-term (5–20 min) treatment with 1 mM taurine had no significant effect on steadystate basal total free Ca2+ and Na+ level ([Ca]i and ([Na]i ). However, short-term exposure to taurine from 5–80 mM induced a significant dose dependent increase of basal [Ca]i and [Na]i of ventricular heart cells. The increase of basal total [Na]i by short-term treatment with taurine (5–80 mM) could be due to Na+ entry through the taurine-Na+ cotransporter [16]. Since taurine had no direct effect on the Na+-Ca2+ exchange current [9], it is thus possible that the increase of basal [Ca]i by short-term treatment with the amino acid could be due to an increase in [Na] i which in turn favours transsarcolemmal Ca2+ influx through Na+-Ca2+ exchange [20, 24]. Using 3-D confocal Ca2+ and Na + measurements, our results show that the increase of basal intracellular free Ca2+ and Na+ by short-term treatment with high concentrations of taurine (≥ 5 mM) are mainly nuclear ([ ]n). The increase of basal [Na]n by short-term treatment with taurine could be due to possible presence of a taurine-Na+ cotransporter in the nuclear membrane. However, the increase of [Ca] n by shortterm treatment with taurine could be due to cytosolic Ca2+ buffering by the nucleus [5, 8]. Our results also show that short-term effect of taurine on [Ca]c and [Na]i is different from the long-term (12–24 h) effect of this amino acid. Long-term treatment with physiologically relevant concentrations (1 mM) of taurine significantly decreased both [Ca]c and [Ca]n as well as [Na]n without affecting [Na]c . The decrease of basal nuclear free Ca2+ and Na+ levels by long-term taurine treatment could be due in part to a decrease in the minimum Ca2+ and Na+ buffering capacity of the nucleus [5, 8], yet the decrease of [Ca]c could be due in part to stimulation of sarcolemma and/or SR Ca2+ pump
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Fig. 4. Effect of long-term exposure to taurine and high extracellular K+ on cytosolic and nuclear free Ca2+ and Na+ in 10-day-old embryonic chick heart cells using Fluo-3 and Sodium Green dyes and three-dimensional laser confocal microscopy. A and F: Resting basal Fluo-3 Ca2+ fluorescence (A) and Sodium Green Na+ fluorescence (F) in the nucleus and cytosol. B and G: Long-term exposure (12–24 h) to 1 mM taurine induced a significant sustained decrease of cytosolic and nuclear free Ca2+ (B) with an apparent decrease of nuclear free Na+(G). C and H: Long-term (12–24 h) depolarization of the cell membrane with 30 mM [K] o induced only a sustained elevation of nuclear free Ca2+ (C) without affecting [Na]c and [Na] n (H). D and I: Depolarization of the cell membrane and addition of 1 mM of taurine (in the presence of high [K] o) for 24 h did not block the increase of nuclear free Ca2+. E and J: Pretreatment with taurine for 12 h and then depolarization of the cell membrane for an additional 12 h (in the presence of taurine) prevents nuclear Ca 2+ overload (E) induced by high [K] o but increased both [Na]n and [Na]c (J). Vertical bars represent means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
activity. Permanent (long-term) depolarization of the cell membrane with high [K]o (30 mM) had no effect on cytosolic free Ca2+ and Na+ as well as on nuclear free Na+. There was however a significant increase of [Ca]n. The absence of effect of permanent depolarization of the cell membrane with high [K] o on cytosolic Ca2+ could be due to cytosolic Ca2+ buffering by the nucleus [5, 8] and transsarcolemmal Ca2+ outflow through the Ca 2+ pump. Long-term pre-treatment with 1 mM taurine followed by long-term depolarization with high extracellular K+ (in presence of taurine) inhibited the increase of [Ca] n induced by depolarization of the cell membrane alone, but significantly increased both [Na]c and [Na] n.
It is possible that the inhibition of the depolarization induced elevation of [Ca]n by pre-treatment with taurine could be due in part to stimulation of transsarcolemmal and nuclear Ca2+ outflow most probably through a Na +-Ca2+ exchanger. It is also possible that long-term pre-treatment with taurine may decrease the resting basal Ca2+ buffering capacity of the nucleus which may in turn mask a possible elevation of [Ca] n induced by later depolarization with high [K] o. The fact that the amino acid did not reverse depolarization-induced Ca2+ overload and that only long-term pre-treatment with taurine blocks the observed elevation of [Ca]n induced by depolarization with high [K]o suggests that taurine possesses a pro-
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Fig. 5. Cross-sectional view of a 3D-reconstructed isolated ventricular myocyte illustrating intracellular Ca2+ distribution in control (A) and in response to long-term pretreatments (24 h) with 1 mM taurine and/or KCl (30 mM) depolarization. Note the decrease of cytosolic and nuclear Ca2+ by long-term exposure to taurine (A vs D). B: long-term (24 h) depolarization of the cell membrane induced increase of [Ca]n. C: long-term depolarization of the cell membrane with long-term treatment with 1 mM taurine did not block the increase of [Ca]n induced with high [K] o (30 mM). Note the high increase of nuclear Ca2+ in (C) when compared to (B). E: Initial pretreatment with 1 mM taurine (12 h) followed by long-term depolarization in presence of taurine prevented the high increase of [Ca]n induced with long-term depolarization only. The white scale bar is in microns. The colour scale on the left represents pseudocolor intensity levels of Ca2+ Fluo-3 dye from 0 to 255. The upper right panels of (A) to (E) represent a 3-D look-through reconstruction of the nucleus (Syto 11 staining) while the lower right panels represent double labelling 3-D reconstruction of free Ca 2+ and the nucleus showing Ca2+ in the cytosol as well as in the nucleus. A, B, C, D and E are different representative cells.
tective effect against Ca2+ overload. A protective effect of taurine against Ca2+ overload as well as against several types of cardiac pathological conditions were suggested previously by several laboratories [2, 22, 24, 26–28]. In summary, these results indicate that short-term treatment with taurine at nearly normal circulating concentrations (1 mM) has no effect on basal Na+ and Ca2+ transport in heart cells. However, long-term treatment, which may mimic a more normal physiological situation, decreases [Ca]c , [Ca]n and [Na]n. Moreover, long-term pre-treatment with taurine seems to inhibit intracellular Ca2+ overload induced by depolarization of the cell membrane which seems to be mainly intranuclear. The mechanisms implicated in taurine action on Na+ and Ca2+ transport through the sarcolemma and nuclear membrane awaits further elucidation.
Acknowledgements This study was supported by Medical Research Council grant no. MT-12882. Dr. Bkaily is a scholar of the Fonds de la Re-
cherche en Santé du Québec. The authors thank Ms Mireille Dussault for her secretarial assistance.
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